US4770997A - Thermostable bilirubin oxidase and production process thereof - Google Patents

Thermostable bilirubin oxidase and production process thereof Download PDF

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Publication number
US4770997A
US4770997A US06/820,011 US82001186A US4770997A US 4770997 A US4770997 A US 4770997A US 82001186 A US82001186 A US 82001186A US 4770997 A US4770997 A US 4770997A
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bilirubin
bilirubin oxidase
oxidase
temperature
reaction
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Eiichi Yoshino
Shigeyuki Imamura
Kazuo Matsuura
Hideo Misaki
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Asahi Kasei Corp
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Toyo Jozo KK
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/03—Oxidoreductases acting on the CH-CH group of donors (1.3) with oxygen as acceptor (1.3.3)
    • C12Y103/03005—Bilirubin oxidase (1.3.3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus
    • Y10S435/836—Bacillus licheniformis

Definitions

  • This invention relates to a novel bilirubin oxidase and to a process for its production. More specifically, this invention relates to novel thermostable bilirubin oxidase and a process for the production of bilirubin oxidase which comprises culturing a bilirubin oxidase producing microorganism belonging to the genus Bacillus and then preparing the resultant bilirubin oxidase from the cultured broth.
  • Bilirubin oxidase has conventionally been known as an enzyme which has substrate specificity to bilirubin and catalyzes a reaction in which biliverdin and water are formed from bilirubin and oxygen.
  • microorganisms capable of producing bilirubin oxidase have been reported certain Myrothecium genus [N. Tanaka and S. Murao, Agric. Biol. Chem., 46, 2499 (1982)], Schizophyllum genus (Japanese Patent Laid-Open No. 135886/1984), and Coprinus, Trametes, Lenzites, Coriolus, Pholiota, Pleurotus and Fomitopsis genus (Japanese Patent Laid-Open No. 198971/1984).
  • Bilirubin oxidase has been attracting interests in recent years as a reagent for determining of bilirubin content and removing bilirubin which causes errors to analysis of other biochemical substance(s) in serum samples in the field of clinical chemistry.
  • the Myrothecium-originated enzyme is stable for 15 minutes as 37° C. but its residual activity is as little as 20% or so at 70° C.
  • the Schizophyllum-originated enzyme is stable for 10 minutes up to 45° C. only.
  • the present inventors have proceeded with a variety of investigations. As a result, it has been found that a microorganism strain, B-0891 strain, isolated from the soil of Owakidani, Hakone-machi,, Ashigarashimo-gun, Kanagawa-ken, Japan and belonging to the genus Bacillus produces bilirubin oxidase capable of catalyzing a reaction in which biliverdin and water are formed from bilirubin and oxygen, and the thus-produced bilirubin oxidase is stable around 70° C.
  • the present invention has been completed based on the above finding, and relates to bilirubin oxidase which have thermostable property and substrate specificity to at least bilirubin and catalyze a reaction in which biliverdin and water are formed from bilirubin and oxygen, and to a process for producing bilirubin oxidase, which comprises culturing a bilirubin oxidase producing microorganism belonging to the genus Bacillus and then preparing the resultant bilirubin oxidase from the cultured broth.
  • the bilirubin oxidase of this invention is excellent in thermostability and hence has very little danger of inactivation or the like upon its immobilization as one way of its enzymatic application or during its handling as a clinical diagnostic. It is therefore easy to handle.
  • the present invention has provided this novel and good bilirubin oxidase.
  • FIG. 1 shows the good thermostability of the bilirubin oxidase of this invention
  • FIG. 2 illustrates the optimum temperature of the bilirubin oxidase of this invention
  • FIG. 3 depicts the good pH stability of the bilirubin oxidase of this invention.
  • FIG. 4 shows the optimum pH of the bilirubin oxidase of this invention.
  • a discrete or short-chain bacillus having round edges Size: 0.5-0.8 ⁇ 2.0-40 ⁇ m. No pleomorphism.
  • Spore shape, size, location, swelling of microorganism body: An oval or cylindrical spore of 0.5 ⁇ 1.0 ⁇ m is formed centrally or at a location close to the center. The microorganism body is not swollen by the spore.
  • Nutrient agar plate culture (28°-30° C., 24 hrs.): A circular, flat and soft colony is formed. The periphery is irregular and the surface is somewhat dry. The color is gray white and no soluble dyestuff is produced.
  • Liquid culture peptone solution, 28°-30° C., 24 hrs.: The growth is weak and a soft-hairy precipitate is developed.
  • BCP milk (28°-30° C., 24 hrs.): The solution is rendered alkaline and is peptionized.
  • the strain of the present invention is a discrete or short-chain, round-edges, gram-positive and non-mobile bacillus microorganism. It is catalase-positive and oxidase-positive and produces acids fermentatively from glucose. It grows at 50° C. and the guanine and cytosine content (GC %) of its nucleric acid is 44%.
  • the present strain was judged to be a strain belonging to the genus Bacillus in view of the above-described principal characteristics thereof.
  • Bacillus licheniformis various characteristics of Bacillus licheniformis were found to be in good conformity with those of the microorganism of this invention. However, some differences were observed in mobility and salt resistance. From the above results, the strain of this invention was judged to belong to Bacillus licheniformis and to be its variant strain. Therefore, the present strain was named as "Bacillus licheniformis B-0891".
  • the above bilirubin oxidase producing microorganism is cultured in a usual manner which is employed to produce enzymes and the like. Its culture may be effected as either liquid culture or solid culture. For industrial production, it is advantageous to inoculate cells of the bilirubin oxidase producing microorganism on a culture medium suitable for use in its production and to subject them to submerged aerated-stirring culture.
  • a culture medium for the culture of bilirubin oxidase may be chosen from a wide variety of culture media which are routinely used for the culture of microorganisms. Its nitrogen source may be any metabolizable nitrogen compound.
  • culturing temperature may be suitably adjusted within a temperature range in which the microorganism is allowed to grow and to produce bilirubin oxidase. Preferably, it is 25°-30° C.
  • the culturing time may somewhat vary depending on conditions. It is usually 7-8 days or so. Needless to say, it is desirable to finish the culture at a suitable time point by watching the time point at which bilirubin oxidase reaches its maximum potency.
  • a usual enzyme-preparing method may be used to purify the enzyme from the cultured broth.
  • the enzyme is found in both cells and cultured broth supernatant, the enzyme is normally obtained, from the viewpoint of the efficiency of its preparation and the like, from the supernatant obtained by removing cells from the cultured broth. It is however possible to prepare the enzyme from the cells by a usual method and to use it in combination with that obtained from the supernatant.
  • the thus-obtained crude enzyme solution is purified by a conventional isolation and purification method which is employed for proteins, enzymes and the like, thereby obtaining bilirubin oxidase in its purified form.
  • a solution containing crude bilirubin oxidase may be subjected to a fractional precipitation method making use of an organic solvent such as acetone, methanol or ethanol or to a salting-out method employing ammonium sulfate, sodium chloride or aluminum sulfate, thereby causing the enzyme to precipitate from the solution and recovering it in a crude form.
  • a fractional precipitation method making use of an organic solvent such as acetone, methanol or ethanol or to a salting-out method employing ammonium sulfate, sodium chloride or aluminum sulfate
  • the precipitate is dissolved in a solvent such as tris-HCl buffer.
  • a solvent such as tris-HCl buffer.
  • the resultant solution is then purified by suitably combining adsorption chromatography, which makes use of an ion-exchange resin such as diethylaminoethylcellulose, diethylaminoethyldextran gel or quaternary aminoethyl-dextran gel, and adsorption chromatography which employs an gel filter aid such as dextran gel or polyacrylamide gel. Thereafter, it is dried by such a technique as lyophilization to obtain bilirubin oxidase in its purified form.
  • a reaction mixture (1.49 ml) of the above composition was placed in a small test tube. After heating it to 37° C., 0.01 ml of a 10 mM bilirubin solution was added to initiate the reaction. They were reacted at 37° C. for 10 minutes. After the reaction, 1.5 ml of 1% CPC (N-cetylpyridinium chloride) was added to terminate the reaction and the reaction mixture was subjected to the colorimetric analysis at 453 nm to determine its absorbance [S]. On the other hand, as a blank, a reaction mixture (1.44 ml) of the same type as the above-employed reaction mixture except for the exclusion of the enzyme solution was added with 0.01 ml of the bilirubin solution.
  • CPC N-cetylpyridinium chloride
  • the resultant mixture was incubated at 37° C. for 10 minutes precisely. Thereafter, 1.5 ml of 1% CPC and 0.05 ml of the bilirubin oxidase solution were added successively. The thus-prepared mixture was subjected to the colorimetric analysis at 453 nm to determine its absorbance [B].
  • the enzymatic activity is calculated in accordance with the following equation: ##EQU1## wherein 1 unit (U) of the enzymatic activity is defined as the amount of enzyme which oxidizes 1 ⁇ mol of bilirubin per minute under the above-described conditions.
  • Results are shown in FIG. 2.
  • the optimum temperature of the bilirubin oxidase of this invention was found to be around 80° C.
  • Portions of the present enzyme were incubated at 37° C. for 1 hour in buffers of various pH levels respectively. Thereafter, their activities were measured by the above-described assay method to determine the influence of pH.
  • Results are shown in FIG. 3, in which , , and indicate acetate buffer (pH 4.0-6.0), dimethylglutaric acid-NaOH buffer (pH 7.5-9.0), phosphate buffer (pH 6.5-8.0) and tris-HCl buffer (pH 7.5-9.0) respectively.
  • the bilirubin oxidase of this invention was found to be stable around pH 7-9.
  • Results are shown in FIG. 3, in which , , and indicate acetate buffer (pH 4.0-6.0), dimethylglutaric acid-NaOH buffer (pH 7.5-9.0), phosphate buffer (pH 6.5-8.0) and tris-HCl buffer (pH 7.5-9.0) respectively.
  • the optimum pH for the bilirubin oxidase of this invention was found to be around pH 8.
  • the bilirubin oxidase of this invention was inhibited by metal ions of Cu 2+ , Zn 2+ and Mn 2+ , CPC and Cation FB (trade name; product of Nippon Oils and Fats Co., Ltd.) and activated by FAD.
  • the isoelectric point was found to be around pH 3.35.
  • the enzyme of this invention is different from any one of the known bilirubin oxidase samples in view of its various enzymochemical and physiochemical characteristics. Pertaining to thermostability, the residual activity after a treatment at 60° C. for 10 minutes was 90-95% even in the case of the bilirubin oxidase originated from Coprinus, which had the best thermostability among the conventional bilirubin oxidase samples, whereas the bilirubin oxidase of this invention remained stable up to 70° C. in the same 10 minute treatment and still showed residual activity as high as 50% even after treated at 90° C. for 10 minutes.
  • the bilirubin oxidase of this invention is considered to be a highly thermostable enzyme.
  • the enzyme of this invention had a very small Km value, i.e., 2.86 ⁇ 10 -5 M and its reactivity was extremely good.
  • Bacillus licheniformis B-0891 (FERM BP-952) was inoculated in a 500 ml Erlenmeyer flask which contained 100 ml of a culture medium (sterilized at 120° C. for 20 minutes; pH 7.5) containing 1.0% (W/V) of sucrose, 1.0% (W/V) of yeast extract ("Meast®", product of Asahi Breweries, Ltd., Tokyo, Japan), 0.3% (W/V of NaCl, 0.05% (W/V) of MgSO 4 .7H 2 O and 0.1% (W/V) of K 2 HPO 4 , and was then cultured with shaking at 30° C. for 24 hrs to obtain a seed culture.
  • the seed culture was inoculated in a jar fermenter having a capacity of 30 l and containing 20 l of a culture medium (sterilized at 120° C. for 20 minutes; pH 7.5; with 0.1% of a defoaming agent "Disfoam CB-442, trade name; product of Nippon Oils & Fats Co., Ltd.” contained therein) having the same composition as the above culture medium and was cultured with aerated stirring at 30° C. and 300 rpm, for 8 days and under aeration conditions of 20 l/min. After completion of the culture, the cultured broth was centrifuged (at 5,000 rpm for 15 minutes) to obtain a supernatant (13.8 l, 200 U).
  • a culture medium sterilized at 120° C. for 20 minutes; pH 7.5; with 0.1% of a defoaming agent "Disfoam CB-442, trade name; product of Nippon Oils & Fats Co., Ltd.” contained therein
  • the supernatant was subjected to ultrafiltration (on a module apparatus) to concentrate it to 1.96 l.
  • the resultant concentrate was then added with chilled acetone in a volume 0.6 times the concentrate, followed by its centrifugation (at 5,000 rpm for 15 minutes) to remove insoluble matter.
  • the thus-prepared concentrate was added with chilled acetone in a volume 1.6 times the concentrate so as to have a bilirubin oxidase active fraction to precipitate.
  • the bilirubin oxidase active fraction was removed by centrifugation (at 5,000 rpm for 15 minutes) and the precipitate was dissolved in 0.2 M tris-HCl buffer to obtain an enzyme solution in a volume of 205 ml (200 U).
  • the enzyme solution was dialyzed overnight against 15 l of 10 mM tris-HCl buffer (pH 7.5). Thereafter, the dialyzate was adsorbed on a DEAE-Sepharose CL-6B column (5.0 ⁇ 14.5 cm) which had in advance bufferized with the above buffer. The column was then subjected to elution by density gradiation with 0-1.0 molar of potassium chloride. Enzyme active fractions eluted over the potassium chloride concentrations of 0.3-0.5 mole were recovered to collect the enzyme of this invention.
  • bilirubin oxidase active fractions were concentrated through an ultrafiltration membrane "XM-50" (trade name; product of Amicon, Corp.), followed by its development with 10 mM tris-HCl buffer (pH 7.5) on a Sephacryl S-200 column (3.6 ⁇ 80 cm). Bilirubin oxidase active fractions over developer fractions of 580-640 ml were recovered and were then lyophilized to obtain 350 mg (90.0 U) of bilirubin oxidase in its powdery form.
  • XM-50 ultrafiltration membrane

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JP60020625A JPH0653069B2 (ja) 1985-02-05 1985-02-05 耐熱性ビリルビン・オキシダーゼの製造法
JP60-20625 1985-02-05

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4985360A (en) * 1987-06-10 1991-01-15 Toyo Jozo Kabushiki Kaisha Novel bilirubin oxidase which has a substrate specifity to bilirubin, but not to biliverdin, catechol and hemin
US5104794A (en) * 1989-03-13 1992-04-14 Unitika Ltd. Quantitative determination of bilirubin and a reagent therefor
US5624811A (en) * 1993-03-24 1997-04-29 Boehringer Mannheim Gmbh Bilirubin oxidase from alfalfa and use of the enzyme
US5945298A (en) * 1997-02-28 1999-08-31 Nitto Boseki Co., Ltd. Method for assaying bilirubin δ fractions
US6190891B1 (en) * 1996-06-06 2001-02-20 Showa Denko K.K. Process for producing high-molecular-weight phenolic compounds with myrothecium
WO2004085640A1 (en) * 2003-03-28 2004-10-07 Council Of Scientific And Industrial Research A process for preparation of thermostable enzyme
US20050209305A1 (en) * 2004-03-19 2005-09-22 Pendrak Michael L Methods for the production of biliverdin
US20080070971A1 (en) * 2006-03-06 2008-03-20 Wang Xiang H Medical Use of Bilirubin and its Structural Analogues
WO2011117839A1 (en) 2010-03-24 2011-09-29 Centre National De La Recherche Scientifique Bacillus pumilus bilirubin oxidase and applications thereof
WO2012160517A1 (en) 2011-05-24 2012-11-29 Centre National De La Recherche Scientifique Bilirubin oxidase from magnaporthe oryzae and applications thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0320095A3 (en) * 1987-12-10 1989-08-02 HEALTH & RESEARCH SERVICES INC. Bilirubin degrading enzymes
US8740994B2 (en) 2011-05-11 2014-06-03 Amano Enzyme Inc. Dyeing agent and use for same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5861000A (ja) * 1981-10-08 1983-04-11 Nippon Shoji Kk 体液成分の酵素的定量におけるビリルピンの干渉除去方法
US4677062A (en) * 1983-04-28 1987-06-30 Kyowa Hakko Kogyo Co., Ltd. Process for producing bilirubin oxidase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5861000A (ja) * 1981-10-08 1983-04-11 Nippon Shoji Kk 体液成分の酵素的定量におけるビリルピンの干渉除去方法
US4677062A (en) * 1983-04-28 1987-06-30 Kyowa Hakko Kogyo Co., Ltd. Process for producing bilirubin oxidase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, vol. 99, No. 3, 18 Juillet 1983, p. 337 resume No. 19274th, Columbus, Ohio, US; & JP A 58 61 000 (Nippon Shoji Co., Ltd) 11 04 1983. *
Chemical Abstracts, vol. 99, No. 3, 18 Juillet 1983, p. 337 resume No. 19274th, Columbus, Ohio, US; & JP-A-58 61 000 (Nippon Shoji Co., Ltd) 11-04-1983.

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4985360A (en) * 1987-06-10 1991-01-15 Toyo Jozo Kabushiki Kaisha Novel bilirubin oxidase which has a substrate specifity to bilirubin, but not to biliverdin, catechol and hemin
US5104794A (en) * 1989-03-13 1992-04-14 Unitika Ltd. Quantitative determination of bilirubin and a reagent therefor
US5624811A (en) * 1993-03-24 1997-04-29 Boehringer Mannheim Gmbh Bilirubin oxidase from alfalfa and use of the enzyme
US6190891B1 (en) * 1996-06-06 2001-02-20 Showa Denko K.K. Process for producing high-molecular-weight phenolic compounds with myrothecium
US6537546B2 (en) * 1996-06-06 2003-03-25 Sds Biotech K.K. Process for macromolecularizing phenolic compounds etc. and use thereof
US5945298A (en) * 1997-02-28 1999-08-31 Nitto Boseki Co., Ltd. Method for assaying bilirubin δ fractions
WO2004085640A1 (en) * 2003-03-28 2004-10-07 Council Of Scientific And Industrial Research A process for preparation of thermostable enzyme
US20050209305A1 (en) * 2004-03-19 2005-09-22 Pendrak Michael L Methods for the production of biliverdin
US7504243B2 (en) 2004-03-19 2009-03-17 The United States Of America As Represented By The Department Of Health And Human Services Methods for the production of biliverdin
US8344019B2 (en) 2004-03-19 2013-01-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Methods for the production of biliverdin
US20090203762A1 (en) * 2005-03-14 2009-08-13 Pendrak Michael L Methods for the production of biliverdin
US20080070971A1 (en) * 2006-03-06 2008-03-20 Wang Xiang H Medical Use of Bilirubin and its Structural Analogues
WO2011117839A1 (en) 2010-03-24 2011-09-29 Centre National De La Recherche Scientifique Bacillus pumilus bilirubin oxidase and applications thereof
WO2012160517A1 (en) 2011-05-24 2012-11-29 Centre National De La Recherche Scientifique Bilirubin oxidase from magnaporthe oryzae and applications thereof
US9260700B2 (en) 2011-05-24 2016-02-16 Centre National De La Recherche Scientifique Bilirubin oxidase from Magnaporthe oryzae and applications thereof

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JPS61209587A (ja) 1986-09-17
IT8647616A0 (it) 1986-02-04
DE3603257A1 (de) 1987-01-08
JPH0653069B2 (ja) 1994-07-20
FR2576909A1 (fr) 1986-08-08
IT1203738B (it) 1989-02-23
FR2576909B1 (fr) 1988-10-14

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